Project Summary Basal cell carcinomas (BCCs) are locally invasive epithelial cancers that affect over 4 million patients a year in the United States and are solely driven by activating mutations in the Hedgehog (HH) pathway. Inappropriate HH pathway activation also drives growth of a variety of cancers including brain, pancreatic, prostate, and small cell lung cancer that account for up to 25% of all human cancer deaths. HH antagonists such as vismodegib are FDA-approved to treat advanced and metastatic BCCs, however, nearly 60% of advanced tumors display inherent vismodegib resistance and 20% of tumors that do respond acquire drug resistance every year. This is a highly relevant issue as advanced BCC cases are estimated to approach 400,000 patients each year, illustrating a critical need to identify therapeutic targets downstream of SMO to suppress HH pathway activity. The consequence of not meeting this need will likely be the inability to treat patients who are resistant to current approved therapies, leading to an increase in mortality for patients inflicted with BCC and other HH-dependent cancers. Our long-term objective is to identify and develop targeted therapeutics to treat drug-resistant HH-driven cancers. The overall objective of this application is to define how GLI is oncogenically activated in HH-driven cancer. Our central hypothesis is that GLI phosphorylation drives transcriptional activation and SMO antagonist-resistant BCC growth, and targeting the signaling pathways that activate GLI will suppress tumor growth. My two specific aims will define 1) how GLI zinc finger phosphorylation and 2) how clinically recurrent GLI mutations promote transcriptional activity, tumor growth, and drug resistance. Defining how GLI is activated in cancer may reveal novel therapeutic targets to treat patients with HH-driven cancers. To achieve these aims, we will use BCC cell lines and allografts that overexpress clinically observed and recurrent GLI mutants to assay for tumor growth in the presence or absence of HH antagonists. We will use the GLI mutants that show increased transcriptional activity to define how GLI is activated in cancer using standard molecular biology, biochemistry, cell biology, and genetic techniques. Our preliminary data has already identified three kinases that regulate GLI activity, and we plan to generate phospho-specific mutants to define when, where, and how each kinase acts on GLI. We will immunofluorescently stain human tumors with appropriate antibodies to verify these pathways operate in humans, and we will perform standard gain- and loss-of-function studies to analyze the pathways involved over the lifetime of the tumor.